We seek to understand at a molecular level the various ways by which an organism maintains the integrity of its genome while accommodating the need for genetic diversity. Our research efforts currently focus on a highly conserved DNA repair pathway, DNA mismatch repair (MMR). MMR, exemplified by the E. coli methyl-directed MMR pathway, targets base pair mismatches that arise through DNA replication errors, homologous recombination and DNA damage. Inactivation of MMR results in a large increase in the rate of spontaneous mutation and is associated with both sporadic and hereditary cancers. We are currently investigating molecular mechanisms of DNA damage signaling mediated by mammalian MMR proteins. SN1 alkylating agents commonly used in chemotherapy produce cytoxic O6-methylguanine residues. Cell killing results from cell cycle arrest and apoptosis triggered by DNA damage recognition by MMR proteins MutSa and MutLa. We are developing an in vitro system in which we can study the molecular mechanism underlying activation of the ATR kinase signaling cascade in response to DNA alkylation. We have shown that MutSa and MutLa recruit ATR to sites of cytoxic O6-methylguanine-thymidine mispairs resulting in activation of the ATR kinase activity and phosphorylation of cell cycle checkpoint proteins such as Chk1 and SMC1. In collaboration with Dr. Dorothy Erie, we are using atomic force microscopy (AFM) to examine the conformations of protein-DNA complexes. Measurement of fractional occupancies at specific locations on a mismatched DNA allowed us to determine the binding affinity, specificity and stoichiometry of MutS bound to mismatched DNAs. These studies reveal a significantly higher affinity for mismatches than was previously obtained by bulk studies of MutS mismatch binding.